Debilitating craniofacial skeletal defects, which occur frequently as a result of congenital disorders, trauma, cancer, and surgical interventions, are some of the most challenging problems for reconstruction. While infants demonstrate the ability to heal large and complex calvarial defects, those older than one year of age have an insufficient healing response to even small skull defects. Although a plethora of strategies have been developed over the past century, the patchwork of methods currently available reflects the inadequacies of each therapeutic technique. In this regard, skeletal stem cell biology holds enormous untapped promise for future tissue engineering applications. The goal of this project is to make autogenous skeletal progenitor cell-based craniofacial skeletal regeneration a clinical reality. Using an interdisciplinary approach, we will bring together our expertise in stem cell biology, bioengineering, and craniofacial surgery to tackle the roadblocks to translation of cell-based skeletal tissue engineering. In Specific Aim 1, we will prospectively isolate pure populations of human skeletal progenitor cells from different tissues and identify key progenitor cell niche factors necessary for their expansion and differentiation. In Specific Aim 2, we will develop a novel high throughput, microfluidics-based FACS (MF-FACS) device for simultaneous isolation and encapsulation of individual skeletal progenitor cells in a hydrogel-based, microenvironment conductive to their regenerative capabilities. In Specific Aim 3, we will assess the regenerative potential of transplanted encapsulated skeletal progenitors in critical-sized calvarial defect models. The role of supplementary niche factors will be further assessed using a tunable macroscale hydrogel scaffolding material. We believe this highly innovative project will ultimately produce an effective cell-based craniofacial skeletal regeneration regiment suitable for clinical testing. PUBLIC HEALTH RELEVANCE: The current tools available to doctors to repair bone structures of the head and face damaged from accidents, birth defects or cancer are inadequate. Using cells that have been removed from the patient, this project seeks to identify, protect, and return only those cells capable of growing into new bone structures. We seek to improve public health by developing a point-of-care method for surgical reconstruction of living bone.